| Assay Method Information | |
| | Activation Assay |
| Description: | In order to make MDA-MB-453 cell become 15000 cells per well, each 36 μl was seeded in Leibovitz's L-15 medium including 10% fetal bovine serum (manufactured by Life Technologies Corp.) in a 384 well plate, followed by culturing at 37° C. overnight in the absence of CO2. The following day, the test compound and DMSO which is a solvent for the test compound as a negative control were diluted with a fresh medium so as to become 10-fold concentration of the final concentration, and the resultant product was added by 4 μl to each well (the test compound had 10 steps in a final concentration from 10000 nM to 0.3 nM, a final concentration of DMSO was 0.1%), followed by culturing at 37° C. for 2 hours in the absence of CO2. After the incubation, 20 μl of a 40% glyoxal solution (Nacalai Tesque) was added to each well, and then cells were fixed by leaving to stand at room temperature for 30 minutes. Thereafter, the supernatant was removed by centrifuging the plate, and then 0.1% Triton X-100-containing Phosphate-Buffered Saline (PBS) was added to each well by 20 followed by incubating at room temperature for 10 minutes. The 0.1% Triton X-100-containing PBS was removed by centrifuging for 8 seconds at 800 rpm (all solution removal operation by centrifuge described below were carried out under the same conditions), and then a blocking solution (ODYSSEY Blocking Buffer; manufactured by Li COR Biosciences) was added to each well by 20 μl, followed by leaving to stand at room temperature for 1 hour. The blocking solution was removed by centrifuging, with the exception of the blocking solution by centrifugation, and then a blocking solution which was diluted such that the amount of phosphorylation antibody (manufactured by Cell Signaling Technology, Inc) of ACC Ser79 as a primary antibody is 1/500 was added to each well by 10 followed by leaving to stand at 4° C. overnight. The following day, the reaction liquid was removed by centrifuging the plate, and then 0.05% Tween-20-containing Tris-Buffered Saline (TBS) (manufacture by Thermo Scientific Inc.; used in 1× in which 20×TBS Tween-20 was diluted with ion-exchange water) was added to each well by 25 followed by washing each well by centrifugal removal. The washing of each well was repeated for a total of 3 times. After washing, a blocking solution which was diluted such that the amount of IRDye 800CW Goat anti-Rabbit IgG (manufactured by Li−CoR Biosciences) as a secondary antibody is 1/1000 was added to each well by 10 μl, followed by leaving to stand at room temperature for 1 hour. After the secondary antibody reaction, the reaction liquid was removed by centrifuging the plate, and then each well was washed three times with 0.05% Tween-20-containing TBS in the same manner as after the primary antibody reaction. After the washing solution was removed, without change, the plate was air-dried at room temperature for 3 hours or longer and signals were measured by Aerius (manufactured by Li−CoR Biosciences). |
| Affinity data for this assay | |
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